초록
Yarrowia lipolytica is one of the most intensively investigated α-ketoglutaric acid (α-KG) producers. A series of studies have been carried out, but enhancing production via the application of metabolic engineering and genetic engineering is associated with potential safety concerns in some countries and regions. In this study, a high-throughput screening procedure was established to solve the problem by an atmospheric and room temperature plasma based random mutagenesis process. This method was implemented based on the use of pH indicators because of the changes in pH during the fermentation process. For the pre-screening procedure, the indicator bromocresol green, which is sensitive to pH but cannot alter the growth of microorganisms on the culture plate, was applied to exclude most of the negative mutants. For the multi-cell-plate screening process, six different pH indicators were tested. Among them, quinaldine red performed best. Based on the high-throughput screening process, a mutant (1-C6) was successfully obtained from 2880 mutants. The titers of α-KG produced by 1-C6 were enhanced by 51.8% and 45.4% compared with those of the wild type in 500-mL shake flasks and a 3-L fermenter, respectively. The high-throughput screening process established here could well facilitate the enhanced production of other similar organic acids by random mutagenesis or evolutionary processes.